Project Summary Pseudomonas aeruginosa (PA) is an opportunistic pathogen that induces a rapidly developing and destructive disease of the cornea and is a global cause of visual impairment and blindness. It is also the most commonly recovered causative organism in contact lens-related disease in developed countries and in ocular trauma/accidents in less developed ones. Of most concern, continued emergence of antibiotic-resistant bacterial strains poses a serious challenge for effective disease management and adjunctive treatments are sorely needed. PA causes 51,000 healthcare associated infections per year in the USA, of which 13 percent are multi-drug resistant (MDR). Developing alternative approaches to antibiotics which are no longer effective against MDR PA is critical and the goal of this application. For this, preliminary in vitro data show resistance patterns for both nonocular and ocular PA MDR clinical isolates and that growth of PA MDR9 (nonocular) isolate is arrested by glycyrrhizin (GLY), an extract of the licorice root. GLY also reduces bacterial viability/biomass, biofilm and adherence (first step in biofilm formation) to mouse corneal epithelial cells. In addition, neither GLY nor the antibiotics [ciprofloxacin, (Cipro), or Meropenem] are toxic to the mouse eye. In vivo preliminary studies using MDR9 show that: GLY is therapeutic (treatment begun 6h p.i.); and that the infected cornea of Cipro +GLY-treated C57BL/6 mice had reduced disease and bacterial plate count when treatment was initiated 18h after infection than Cipro alone. Based on preliminary data, we hypothesize that GLY with antibiotic is a better regulator of virulence factors in MDR PA (non-ocular and keratitis) isolates and bioenhances the effects of antibiotics. Our overall objective is to test the effects of GLY on killing MDR (nonocular and keratitis) PA, its ability to bioenhance antibiotics to which the isolates are resistant and to do this by determining mechanisms of action against bacterial virulence factors in vitro and in innate immunity in vivo (mouse model of keratitis). We expect to identify additional bacterial and host targets for therapeutic development and the interplay of bacterial and host factors affected by treatment. Two aims are proposed: Specific Aim 1: Tests the hypothesis that GLY kills MDR isolates and enhances antibiotic efficacy by regulating bacterial virulence factors/antibiotic resistance mechanisms: bacterial membrane permeability, efflux of small molecules, biofilm formation/adherence, and MIC and time kill in MDR nonocular and keratitis PA clinical isolates. In this aim we will test the effect of GLY on membrane permeability using flow cytometric quantification of propidium iodide, assay for efflux activity by ethidium bromide (EB) staining and flow cytometry, determine biofilm formation inhibition activity using crystal violet and adherence assays and effect on antibiotics (MIC and time kill). Correlative proteomics to determine the effects of antibiotics +/-GLY on these virulence factors also will be done. Specific Aim 2: Tests the hypothesis that GLY will bioenhance and improve antibiotic killing in corneal infection induced by MDR PA by regulation of host innate immune responses. In this aim, we will test the host innate immune mechanisms which are altered by GLY enhancement of antibiotic killing when treatment is initiated 18h after disease onset. We predict increased effective regulation of innate immunity, reduction in plate count and cellular infiltrate and identification of novel host molecules which will provide additional host treatment targets for keratitis.